Journal of General Microbiology (1992), 138, 1379-1 386. Printed in Great Britain
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Isolation and characterization of a conjugative plasmid from Legionella pneumophila CLIFFORD S. MINTZ,~* BARRYS. FIELDS^ and CHANG-HUA Zou' Department of Microbiology and Immunology, University of Miami School of Medicine, Miami, Florida 33I0 I , USA Respiratory Diseases Branch, Division of Bacterial and Mycotic Diseases, National Center for Infectious Diseases, Centers for Disease Control, Atlanta, Georgia 30333, USA (Received 2 January 1992; revised 2 March 1992; accepted I1 March 1992)
The conjugative properties of an indigenous 85 MDa plasmid (designated pCH1) from Legioneffapneumophifa were studied. To determine if pCHl was transmissible by conjugation, mating experiments were brformed between legionellaethat harboured pCHl and several plasmid-lessrecipients. Plasmid transfer was monitored by colony hybridization, using a cloned 21.0 kb SalI restriction fragment from pCHl as a probe. The results from these experiments showed that pCHl could be conjugatively transferred into several strains of L. pneumophifa serogroup 1 but not into strain Bloomington-2 (serogroup 3) or Escherichia cofi. Southern hybridization experiments in which pCHl DNA was used as a probe showed that pCHl does not share homology with other indigenousL.pneumophifaplasmids. There was no detectable DNA homology between pCHl and L.pneumophifa chromosomal DNA. Additional mating experiments revealed that pCHl was unable to mobilize the L. pneumophifachromosome. The conjugative transfer of pCHl into plasmid-less avirulent or virulent serogroup 1strains did not alter the intracellular growth characteristicsof these strains in U937 cells, a human-monocytelike cell line, or in the amoeba Hartmanneffavermiformis.These results suggest that pCHl does not contribute to the ability of L. pneumophifato enter or grow within eukaryotic cells.
Int d u c tion Legionella pneumophila, the causative agent of Legionnaires' disease, is a facultative intracellular pathogen capable of entering and multiplying in cultured animal cells (Dreyfus, 1987; Pearlman et al., 1988), free-living amoebae (Fields et al., 1986; Holden et al., 1984; Rowbotham, 1986; Newsome et al., 1985) and human mononuclear phagocytes (Horwitz & Silverstein, 1980). At the present time, little is known about the bacterial factors that promote the intracellular lifestyle of this organism. Plasmids have been reported to carry genes that contribute to the virulence of a variety of Gram-negative intracellular pathogens including Yersinia spp. (BenGurion & Shafferman, 1981; Portnoy et al., 1981, 1984), Shigella spp. (Maurelli & Sansonetti, 1988) and Salmonella spp. (Gulig, 1990). Several groups have identified plasmids in both clinical and environmental isolates of
* Author for correspondence.Tel. (305) 547 6310; fax (305) 548 4623. Abbreviation: mAb, monoclonal antibody. 0001-7315 0 1992 SGM
L. pneumophila (Johnson & Schalla, 1982; Knudson & Mikesell, 1980; Maher et al., 1983; Mikesell et al., 1981). Early work indicated that the plasmids ranged in size from 23-85 MDa and did not confer an identifiable phenotype on strains that harboured them (Johnson & Schalla, 1982; Maher et al., 1983). Despite the presence of these plasmids in L. pneumophila, their contribution to the ability of L. pneumophila to enter and grow within eukaroytic cells has never been evaluated. Previous work from this laboratory (Mintz & Shuman, 1988) using broad host range IncP and IncQ plasmids showed that conjugation is possible in L. pneumophila. This finding, along with the identification of an 85 MDa plasmid in numerous L. pneumophila serogroup 1 clinical and environmental isolates (Schalla & Johnson, 1982; Maher et al., 1983) suggested that certain indigenous L. pneumophila plasmids may be transmissible by conjugation. In the present study, we evaluated the conjugative and virulence properties of an 85 MDa plasmid (designated pCH1) from L. pneumophila. Our results demonstrate that pCH 1 is self-transmissible by conjugation among serogroup 1 isolates of L. pneumophila. Also, pCHl does
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Table 1. Bacterial strains Abbreviations: Kmr, kanamycin resistant; Nal', naladixic acid resistant; Rip, rifampicin resistant; Sm', streptomycin resistant ; Trp-, tryptophan auxotroph; r-m+, restriction minus modification plus; r-m-, restriction minus modification minus. Designation L. pneumophila Philadelphia-1 Pontiac-1 NY 26 Ver 5 Rockport CH- 1 RH-1 UH-2 UPH-1 OLDA LPP2 LPP3 SF3256 SF3257 Albuquerque-1 Atlanta-1 Atlanta-4 AM511 CS267 CZ3001 Bloomington-2
cs2
CS302 Knoxville-1 CS323 E. coli DH5a
Relevant characteristics
Comments
Wild-type, clinical serogroup 1 isolate Clinical serogroup 1 isolate Clinical serogroup 1 isolate Clinical serogroup 1 isolate Clinical serogroup 4 isolate Environmental serogroup 1 isolate Environmental serogroup 1 isolate Environmental serogroup 1 isolate Environmental serogroup 1 isolate Clinical serogroup 1 isolate Environmental serogroup 1 isolate Environmental serogroup 1 isolate Environmental serogroup 1 isolate Environmental serogroup 1 isolate Clinical serogroup 1 isolate Clinical serogroup 1 isolate Clinical serogroup 1 isolate Smrr- m+ SmrRiPr-m+ AM511 : :TnSmob KMrSmr Clinical serogroup 3 isolate Serogroup 3 Smrt-m+ Serogroup 3 Sm'RiP Trp r-m+ Avirulent serogroup 1 clinical isolate Serogroup 1, SmrRiP
Plasmid-less Plasmid-less Plasmid-less Plasmid-less Plasmid-less Contains an 85 MDa plasmid Contains 45 and 85 MDa plasmids Contains a 45 MDa plasmid Contains a 33 MDa plasmid Contains an 85 MDa plasmid Contains an 85 MDa plasmid Contains 45 and 85 MDa plasmids Contains an 85 MDa plasmid Contains an 85 MDa plasmid Contains an 85 MDa plasmid Contains a 62 MDa plasmid Contains a 24 MDa plasmid Derived from Philadelphia-1 Spontaneous RiP mutant of AMSII Plasmid-less Plasmid-less, spontaneous Smr mutant of Bloomington-2 Derived from CS2 Plasmid-less Derived from Knoxville-1
NaP r- m+
Source*
H. Horwitz R. Miller M. Horowitz
1
J. F. Plouffe M. Horwitz
R. Miller J
A. Marra This study This study M. Horowitz This study This study R. Miller This study
C. Collins
* Addresses: M. Horwitz, UCLA Medical School, Los Angeles,CA, USA ;R. Miller, Universityof LouisvilleSchool of Medicine, Louisville, KY, USA; J. F. Plouffe, Ohio State University, Columbus, OH, USA; A. Marra, Massachusetts Institute of Technology, Boston, MA, USA; C. Collins, University of Miami School of Medicine, Miami, FL, USA. not contribute to the ability of L. pneumophila to enter or grow within eukaryotic cells.
Methods Bacterial strains and growth media. The bacterial strains used in this study are listed in Table 1. L. pneumophila was routinely grown in Albumin Yeast Extract (AYE) broth and on ACES-buffered Charcoal Yeast Extract (ABCYE) agar plates at 37 "C as previously described (Mintz & Shuman, 1988). Strains of Escherichia coliwere grown at 37 "C on L-agar or in L-broth. When necessary, the following antibiotic concentrations were used : streptomycin (Sm), 50 pg m1-I ; rifampicin (Rif), 20 pg ml-l; kanamycin (Km), 25 pg ml-l; ampicillin (Ap), 50 pg ml-l and tetracycline (Tc), 10 pg ml-l. Isolation and analysis of plasmid DNA. Plasmid DNA was isolated from L. pneumophila using the rapid method of Kado & Liu (1981) or the alkaline lysis protocol of Birnboim & Doly (1979). In some experiments, chromosomal DNA was removed from plasmid-containing lysates by centrifugationthrough ethidium bromide/CsClgradients (Maniatis et al. 1982). Plasmid DNA prepared by the Kado & Liu method was not cut by restriction endonucleases so plasmid DNA isolated by the Birnboim & Doly method was used for restriction endonuclease analysis in this investigation. Plasmid DNA and restriction enzyme digestions of plasmid DNA were analysed by
horizontal agarose gel electrophoresis using 0.4% and 0.7% agarose (Bio-Rad) gels. CloningofpCHl DNA. CsC1-purified pCHl DNA was digested with the restriction enzyme SalI (Promega), ligated with SalI-cleaved, alkaline-phosphatase-treated pBR322 DNA and transformed into CaC1,-treated E. coli DH5a. Transformants were selected on L-agar supplemented with ampicillin and putative recombinants were identified by their AprTcs phenotypes. The presence of pCHl sequences in recombinant plasmids was confirmed by Southern hybridization using 32P-labelledpCHl DNA as a probe. Mating experiments. Donor strain CH-1 (which harbours pCH 1 and contains no antibiotic-resistance markers) and antibiotic-resistant plasmidless recipients were grown to early exponential phase in AYE broth at 37 "C. Equal numbers of donors and recipients were incubated together on non-selectiveABCYE plates at 37 "C for 18 h as described by Mintz & Schuman (1988). After incubation, cells were removed from the plates, serially diluted in M63 salts buffer (Silhavy et al., 1984) and spread onto ABCYE agar plates that contained appropriate antibiotics (Sm, Rif or Km) to counterselect the donor strain CH-1. In some matings, strains that contained two or more drug markers were used as recipients. In these experiments, ABCYE agar that contained two antibiotics was used to counterselect CH-1. The plates were incubated at 37 "C. Colonies were replica-plated onto nitrocellulose filters, lysed with NaOH, probed with 32P-labelledpBR322 DNA (containing a 21.0 kb SalI fragment from pCH1) and subjected to
Conjugativeplasmid from Legwnella pneumophila autoradiography. We routinely screened approximately 2000-3000 transconjugants for the presence of pCHl DNA. Preliminary experiments showed that 32P-labelled pBR322 DNA by itself did not hybridize with pCHl or L. pneumophila chromosomal DNA. Hybridizations were performed under conditions of high stringency (Silhavy et al., 1984). Colonies that hybridized with the probe were picked from master plates and purified twice by passage on selective media. Plasmid DNA was isolated from these colonies, digested with SalI and examined by agarose gel electrophoresis. All mating experiments were repeated at least two times. For mating experiments involving DNAase treatment, equal numbers of donors and recipients were incubated for 18 h at 37 "C on ABCYE agar that contained 1 mg DNAase ml-l. Cells were then removed from the plates and spread onto antibiotic-containing ABCYE agar as described above. Chromosome mobilization experiments using strain CH-1 as a donor and auxotrophic mutants of strains Bloomington-2 (Gua- and Trp-) and Philadelphia-1 (Thy- and Trp-) as recipients were done according to Mintz & Schuman (1988). Colony immunoblast assay. Single colonies of donors, recipients and transconjugants were inoculated onto 0.45 pm nitrocellulose filters (Fisher) placed on ABCYE agar plates and incubated for 24-48 h at 37 "C. Filters were removed from the plates and incubated in Trisbuffered saline (TBS; 50 mM-Tris/HCl, 150 mM-NaCl, pH 7.5) containing 5 % (w/v) nonfat dried milk (Carnation) for 2 h at room temperature. The filters were washed several times with TBS and incubated with monoclonal antibody (mAb) 1E6 (1 : 1000dilution) on a rotary shaker (New Brunswick) for 1 h at room temperature. mAb 1E6 (kindly provided by W. Johnson, University of Iowa, USA) is specific for serogroup 1 lipopolysaccharide. After incubation, the filters were washed several times with TBS and probed with horseradishperoxidase-conjugated goat-antimouse IgG antibodies (1 : 1000 dilution, Cappel) for 1 h at room temperature. After a series of four washes with TBS, filters were immersed in a solution of 0.05% 4-chloro-lnaphthol (Sigma) and 0.015 % hydrogen peroxide. Antibiotic and heavy metal resistance. The ability of pCH 1 to confer resistance to antibiotics or heavy metals was tested in the following manner. Single colonies of isogenic plasmidless and pCH 1-containing strains of L. pneumophila were inoculated onto ABCYE agar and ABCYE agar containing a specific antibiotic or heavy metal. The plates were incubated at 37 "C for 5 d. Resistance to the following heavy metals was tested using the concentrations suggested by Trevors et al. (1985): cadmium chloride (1 m~), sodium arsenate (38 mM), cobalt chloride (3 mM), copper sulphate (20 mM), lead nitrate (0.3 mM), nickel chloride (3 mM), zinc chloride (1 mM) and silver nitrate (5 mM). Antibiotics were tested at the concentrations mentioned above. Infection of U937 cells and Hartmannella vermiformis with L. pneumophila. Human-monocyte-like cells (U937) were infected with legionellae as previously described (King et al., 1991). U937 cell monolayers were grown in RPMI 1640 (Cellgro) plus 20% (v/v) normal human serum for up to 3 d at 37 "C in 5 % (v/v) C02.At daily intervals, samples were removed from the infected monolayers, diluted in M63 salts buffer and plated on ABCYE agar to determine numbers of L. pneumophila. Plate-grown legionellae were co-cultured with the amoeba H . vermiformis at 35 "C for 7 d according to King et al. (1991). At various times, samples were removed from the co-cultures, serially diluted in buffer, and plated on ABCYE agar to determine the numbers of L. pneumophila. Previous studies using U937 cells (King et al., 1991; Pearlman et al., 1988) and H. vermiformis (King et al., 1991) demonstrated that any increase in numbers of L. pneumophila as measured above represent intracellular growth of the bacterium.
138 1
Isolation and characterization of pCHl The plasmid content of five serogroup 1 strains (CH-1, OLDA, Albuquerque 1, LpP, and SF3256) reported to carry 85 MDa plasmids was analysed by agarose gel electrophoresis. Each strain contained a single plasmid and the electrophoretic mobility of the plasmids was identical (data not shown). Digestion of each plasmid with SalI or BamHI yielded identical fragment patterns (data not shown). These results suggested that strains CH-1, OLDA, Albuquerque-1, LpP, and SF3256 harboured the same plasmid, subsequently designated pCH1. Restriction endonuclease analysis of pCH 1 revealed that EcoRI, Hind111or HaeIII generated 2 13 restriction fragments. Digestion with BglI, PvuII, SalI, BglII or NheI yielded 5-7 restriction fragments (data not shown). pCH1 was not cleaved by NotI, SI', ApaI or RsrII. We also determined that pCHl has a single BarnHI site contained within the 11 kb SalI restriction fragment (Fig. 1). Attempts to construct a detailed restriction map of pCHl were hampered by the large number of restriction fragments generated during single and double digests and our inability to clone the majority of the plasmid genome in E. coli (see below). Pulse-field gel electrophoresis of BamHI-digested plasmid DNA showed that the apparent molecular size of pCH 1 is 125130 kb (data not shown). This is in agreement with the predicted molecular size of pCH1, which was previously determined to be 85 MDa (128 kb). The results from antibiotic sensitivity tests indicated that pCH1 did not encode resistance to Rif, Sm, Ap, Km or Tc. Neither did the plasmid confer resistance to any of the heavy metals tested. Attempts to cure pCH1 from plasmid-bearing strains by treatment with acridine orange (Riva et al., 1973), ethidium bromide (Bouanchaud et al., 1969), growth at 41 "C or by repeated subculture on ABCYE agar at 37 "C were unsuccessful. This suggested that pCH1 was stably maintained in L. pneumophila in the absence of obvious selective pressure. Cloning of pCHl DNA As shown in Fig. 1, the restriction enzyme SalI cleaves pCH1 DNA into six distinct restriction fragments. We attempted to isolate and clone each of the six SalI fragments from pCH1. Despite repeated attempts using either pBR322 or pACYC184 as cloning vectors, we were only able to isolate the 21.0 kb SalI fragment from pCH1. This fragment was cloned into the unique SalI site of pBR322 (Fig. 1) and subsequently used as a probe in plasmid transfer experiments.
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Fig. 1 . Ethidium-bromide-stained 0.4% agarose gel that contains the cloned 21.0 kb SalI fragment of pCH1. Lanes : A, 1 Hind111 molecular size standards; B, undigested pCHl DNA; C, pCHl DNA digested with SalI; D, pBR322 DNA that contains the cloned fragment digested with SalI; E, pBR322 DNA digested with SalI. Arrow indicates the position of the 21.0 kb fragment of pCH1.
Conjugative transfer of pCHl
To determine whether pCHl is conjugative, mating experiments between strain CH-1 (which harboured pCH 1) and several antibiotic-resistant plasmid-less L. pneumophila recipients were done. Since pCHl does not encode any selectable markers, we assessed the ability of strain CH-1 to conjugally transfer pCH1 by colony hybridization using the cloned 21 kb SalI fragment from pCHl as a probe. Initial mating experiments, in which strain AM51 1 was used as the recipient, revealed that several colonies on the selection plates hybridized with the pCHl probe. The electrophoretic profile of SalI-digested plasmid DNA from each of these colonies was identical to that of SalI-digested pCH1 DNA (data not shown). This suggested that pCH1 was transferred by a mating
process. However, the possibility existed that colonies that contained pCH 1 were spontaneous antibioticresistant mutants of strain CH-1 rather than actual transconjugants. To eliminate this possibility, we did additional mating experiments using multiple-antibioticresistant recipients in which two antibiotics were used to counterselect donor strain CH- 1. The frequency of spontaneous mutation to resistance for two antibiotics for strain CH-1 would be approximately lo-' *. However, pCH 1 was detected in exconjugants at frequencies ranging from 10-3-10-4 per recipient. These results supported the idea that colonies that contained pCHl were transconjugants rat her than anti biotic-resistant mutants of strain CH-1. This was confirmed by colony immunoblot experiments with putative transconjugants using mAb 1E6, which does not bind to strain CH-1 but is reactive with each of the other serogroup 1 strains used as recipients in this study. In all cases, colonies that hybridized with the pCHl probe also bound mAb lE6 (data not shown). This demonstrated that pCHl was transferred from strain CH-1 to L. pneumophila recipients during the mating process. The detection of pCH 1 in transconjugants obtained from matings in the presence of DNAase (1 mg ml-I) provided conclusive evidence that pCH l was selftransmissible by conjugation (data not shown). A summary of the mating experiments done in this study is presented in Table 2. By determining the number of pCH1-containing colonies among the total number of transconjugants screened, we estimated that pCH 1 was transferred at frequencies ranging from l 0-3- 10-4 per recipient. Of interest, pCHl could only be conjugally transferred into serogroup 1 recipients. We were unable to detect transfer of pCHl into a serogroup 3 recipient (strain Bloomington-2) or E. coli DHSa. Additional mating experiments between strain CH- 1 and several serogroup 1 or serogroup 3 auxotrophic recipients showed that pCH1 could not promote the chromosomal transfer of the gua, thy or trp loci in L. pneumophila (data not shown). pCHI does not share homology with L. pneumophila chromosomal DNA or other indigenous plasmids
To determine if pCH1 shared DNA homology with other L. pneumophila plasmids or with L. pneumophila chromosomal DNA, we performed colony hybridization experiments with plasmid-less and plasmid-containing strains of L. pneumophila. In these experiments, CsC1-purified pCH1 DNA labelled with 32Pwas used as a probe. pCH1 DNA did not hybridize with strains of L. pneumophila that contained indigenous plasmids other than pCHl (strains UH-2, UPH-1, Atlanta4 and Atlanta-4; Fig. 2). This suggested that pCH1 does not share detectable
Conjugative plasmid from Legionella pneumophila
I 38 3
Fig. 2. Colony hybridization experiments with plasmid-less and plasmid-bearing strains of L. pneumophila. Individual colonies from plasmid-lessand plasmid-bearing strains of L.pneumophila were patched onto nitrocellulosefilters,treated with NaOH and probed with 32P-labeHedCsC1-purified pCHl DNA. Colonies that hybridized with the probe were visualized by autoradiography. Only those strains that harboured an 85 MDa plasmid hybridized with the pCHl probe.
Table 2. Summar? of mating experiments Abbreviations, see Table 1. Recipient Donor
Strain
CH-1 CH-1 CH-1 CH-I CH-1 CH-1 CH-1
AM51 1 CS267 CZ3001 CS320 CS2 CS302 DH5a
Phenotype
Selection
Plasmid transfer detected
Philadelphia-1 serogroup 1 Smrr-m+ Philadelphia-1 serogroup 1 SmrRiPr-m+ Philadelphia-1 serogroup 1 : :Tn5mb KmrSmrr-m+ Knoxville-I serogroup 1 Srn'RiP Bloomington-2 serogroup 3 Sm'r-mBloomington-2 serogroup 3 RiP Trp-r-mEscherichia coli Nalrr-m+
Sm Sm Rif Km Sm Sm Rif Sm Rif Nal
Yes Yes Yes Yes No No No
DNA homology with other Legionella plasmids. In contrast, all of the L. pneumphila strains that contained an 85 MDa plasmid hybridized with the probe (LpP2, LpP,, OLDA, RH-1, SF3256 and SF3257; Fig. 2).
The plasmid-less serogroup 1 strains Philadelphia-1, Knoxville-1 and UH-1 did not hybridize with pCH1 DNA (Fig. 2). Also, the plasmid-less serogroup 3 strain, Bloomington-2, did not hybridize with pCH 1.
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1
C. S . Mintz, B. S . Fields & C.-H. Zou
.,
0
1
2
1 3 0 Time post-infection (d)
1
2L
Fig. 3. Infection of U937 cell monolayers with isogenic plasmid-less and plasmid-bearing strains of L. pneumophila. U937 cell monolayers were infected with L. pneumophila according to the methods of King etul. (1991). (a) AM51 1 ; 0 ,AM51 l(pCH1). @)A,CS323; A, CS323(pCHl). Each point represents the mean f SE for three separate U937 cell cultures.
Additional experiments demonstrated that pCH 1 DNA also did not hybridize with the plasmid-less serogroup 1 strains Pontiac-1, NY 26 and Ver 5, or the plasmid-less serogroup 4 strain Rockport (data now shown). This suggests that these strains do not contain chromosomal sequences homologous with pCHl DNA. pCHl does not aflect the intracellular growth of L . pneumophila
Using the mating procedure outlined above, we introduced pCHl into strains AM51 1 and CS323 to construct isogenic pairs of plasmid-less and plasmidcontaining strains. Strain AM5 11 is virulent and capable of intracellular growth (Marra & Shuman, 1989). In contrast, strain CS323 is avirulent and incapable of intracellular multiplication (C. S. Mintz, unpublished). Both isogenic sets were used to infect monocyte-like U937 cells or the amoeba Hartmannella vermiformis. pCHl did not affect the ability of strain AM511 to multiply in U937 cells (Fig. 3a) or in H. vermiformis(data not shown). Moreover, the plasmid did not restore the ability of the avirulent strain CS323 to replicate in either U937 cells (Fig. 3b) or in H. vermiformis (data not shown).
Discussion In this study, we have shown that pCH1, an indigenous L. pneumophila plasmid, is self-transmissibleby conjugation among serogroup l strains of L . pneumophila. Prior
to the present study, we (Mintz & Shuman, 1988) as well as others (Dreyfus & Iglewski, 1985; Chen et al., 1986) demonstrated that broad host range plasmids of the IncP and IncQ incompatibility groups could easily be transferred via conjugation between different L. pneumophila isolates. Recently, Tully (1991) demonstrated that a 36MDa plasmid from L . pneumophila strain Dodge (serogroup 1) could be transferred by conjugation to other serogroup 1 strains. Our results, along with those of Tully (199I), suggest that conjugation mediated by indigenous plasmids may be a mechanism of genetic exchange for L. pneumophila. To date, neither transformation nor transduction has been reported for L. pneumophila. Despite repeated attempts, we were unable to detect conjugative transfer of pCHl into a serogroup 3 strain (Bloomington-2) of L. pneumophila. This was somewhat surprising since strain Bloomington-2 lacks the LpnII restriction-modification system of L . pneumophila (Marra & Shuman, 1989), and is an excellent recipient in homologous and heterospecific matings involving IncP and IncQ plasmids (Chen et al., 1984; Mintz & Shuman, 1988). It is not clear why we were unable to detect transfer of pCHl in strain Bloomington-2. It is possible that pCH1 cannot be stably maintained in this strain. Experiments are underway to determine if pCH1 can be transferred by conjugation into strains from other serogroups of L . pneumophila. pCHl could not be transferred from strain CH-1 into E. coli strain DH5a. Since the 36 MDa plasmid described by Tully (1991) could also not be transferred from serogroup 1 strains of L. pneumophila into E. coli or Pseudomonas aeruginosa, it would seem that conjugative L . pneumophila serogroup 1 plasmids have a relatively narrow host range. Colony hybridization experiments revealed that pCH 1 did not share detectable DNA homology with the 24,33, 45 or 62 MDa plasmids harboured by certain serogroup 1 strains of L. pneumophila, and that pCHl was not related to these other L. pneumophila plasmids (Fig. 2). Therefore, it is likely that pCHl belongs to a different incompatibility group than these other L . pneumophila plasmids. In support of this idea, the 45 MDa plasmid has been found in two serogroup 1 strains that also harbour pCH1, viz. RH-1 (Maher et al. 1983) and LpP3 (C. S. Mintz, unpublished). Attempts to assign pCHl to a known incompatibility group have been hampered by the lack of a selective marker on pCH 1 and the difficulty associated with introducing plasmid DNA into L. pneumophila. Recently, we determined that electroporation is an efficent way of delivering plasmid DNA into L. pneumophila (C. S. Mintz & C.-H. Zou, unpublished). This should permit future experiments designed to identify the incompatibility group of pCHl. It is well-establishedthat the transfer of chromosomal
Conjugative plasmid from Legionella pneumophila
genes during conjugation results from integration of a conjugative plasmid, such as F, into the donor chromosome (Deich & Green, 1987; Silhavy et al., 1984). Integration of plasmid DNA into the host chromosome usually occurs by recombination between homologous DNA sequences contained on the plasmid and the chromosome (Bartowsky et al., 1987; Brenton et al., 1985). As previously noted, several plasmid-less L. pneumophila strains failed to hybridize with the pCH1 probe in colony hybridization experiments. These results indicate that there is no detectable DNA homology between the L. pneumophila chromosome and pCH1. This finding could explain the inability of pCH1 to mobilize the L. pneumophila chromosome. In the absence of DNA sequence homology, pCH1 would be unable to integrate into the L. pneumophila chromosome. Consequently, the unintegrated plasmid would be incapable of promoting the transfer of chromosomal markers. Of interest, Bartowsky et al. (1987) suggested that the lack of chromosome mobilizing activity exhibited by a conjugative plasmid from Vibrio cholerae was due to the lack of significant homology between the V . cholerae chromosome and the plasmid. Thus, although pCHl is selftransmissible by conjugation, it is not capable of promoting detectable chromosomal gene transfer in L. pneumophila. Virulence plasmids are essential for the pathogenicity of a variety of intracellular pathogens including Salmonella spp. (Gulig, 1990), Shigella spp. (Maurelli & Sansonetti, 1988) and Yersinia spp. (Portnoy et al., 1981, 1984). In the light of these observations, we evaluated the contribution of pCHl to the ability of L. pneumophila to multiply intracellularly in eukaryotic cells. The introduction of pCH1 into the plasmid-less, virulent strain AM51 1 did not augment or alter the ability of this strain to grow within U937 cells or H. vermiformis. Moreover, the presence of pCHl in the avirulent strain CS323 did not restore its ability to multiply in U937 cells or H. vermiformis. These results indicate that pCHl does not encode factors thzt contribute to the intracellular growth of L. pneumophila within eukaryotic host cells. Our results are consistent with the findings of Bollin et al. (1985) who compared the ability of a plasmid-less and plasmid-containing serogroup 1 strain to infect guinea pigs following intreperitoneal injection. The plasmidcontaining strain harboured pCH1 and a 45 MDa plasmid. Their results showed that there was no statistically significant difference in the IDs0 of the plasmid-less or plasmid-bearing strain. Interestingly, the LDS0of the plasmid-less strain was significantly lower (approximately 10-fold) than that of the plasmidcontaining strain. However, it is important to note that the plasmid-less and plasmid-containing strains used in this study were not isogenic. Therefore, it is difficult to
1385
attribute the reduced virulence exhibited by the plasmidcontaining strain to the presence of pCHl or the 45 MDa plasmid. Nonetheless, our results suggest that pCH1 is not required for expression of virulence by L. pneumophila. Although pCHl cannot mobilize the L. pneumophila chromosome,the self-transmissiblenature of the plasmid suggests that it may contribute to the exchange of genetic information among serogroup 1 isolates. For example, the acquisition by pCHl of transposons which code for antibiotic resistance could pose serious problems in the treatment of Legionnaires’ disease. Moreover, the presence of transposons on pCH 1 could facilitate integration of the plasmid into the L. pneurnophila chromosome by transposon-mediated recombination (Ichige et al., 1989, Pischl & Farrand, 1983). This, in turn, could promote Hfr-like chromosomal gene transfer in L. pneumophila. In support of this notion, we have previously demonstrated that conjugative plasmids that contain transposons can be used to mobilize the L. pneumophila chromosome (Mintz & Shuman, 1988). At the present time, it is not clear what functions are encoded by pCH 1. It does not encode resistance to any of the antibiotics or heavy metals tested nor does it contribute to the intracellular growth of L. pneumophila. The absence of pCHl in many environmental and clinical serogroup 1 isolates suggests that any function(s) encoded by the plasmid are not essential for the survival of L. pneumophila in aquatic environments or in the infected host. We thank M. Horwitz, R. Miller, J. Plouffe and C. Collins for supplying us with strains used in this study. This work was supported by a research grant awarded to C. Mintz from the American Lung Association of Florida.
References BARTOWSKY, E. J., MORELLI, G., KAMKE, M. & MANNING, P. (1987). Characterization and restriction analysis of the P sex factor and the cryptic plasmid of Vibrio cholerae strain V58. Plasmid 18, 1-7. BEN-GURION,R. & SHAFFERMAN, A. (1981). Essential virulence determinants of different Yersinia species are carried on a common plasmid. Plasmid 5, 183-187. BIRNBOIM, H. C. & DOLY,J. (1979). A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Research 7 , 1513-1523. BOLLIN,C. E., PLOUFFE, J. F., PARA,M. F. & PRIOR,R. B. (1985). Difference in virulence of environmental isolates of Legionella pneumophila. Journal of Clinical Microbiology 21, 674-677. BOUANCHAUD, D. H., SCAVIZZI, M. R. & CHABBERT, Y. A. (1969). Elimination by ethidium bromide of antibiotic resistance in enterobacteria and staphylococci.Journal of General Microbiology 54, 417-425. BRENTON, A. M., JAOUA, S. & GUESPIN-MICHEL, J. (1985). Transfer of plasmid RP4 to Myxococnts xanthus and evidence for its integration into the chromosome. Journal of Bacteriology 161, 523-528.
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CHEN,G. C. C., LEMA,M. & BROWN,A. (1984). Plasmid transfer into members of the Legionellaceae. Journal of Infectious Diseases 150, 5 13-5 16. CHEN, G. C. C., BROWN,A. & LEU, M. W. (1986). Restriction endonuclease activities in the legionellae. Canadian Journal of Microbiology 32, 591-593. DEICH,R. A. & GREEN,B. A. (1987). Mobilization of Haemophilus infuenzae chromosomal markers by an Escherichia coli F factor. Journal of Bacteriology 169, 1905-1910. L. A. (1987). Virulence associated ingestion of Legionella DREYFUS, pneumophila by HeLa cells. Microbial Pathogenesis 3, 45-52. DREYFUS,L. A. & IGLEWSKI,B. H. (1985). Conjugation-mediated genetic exchange in Legwnella pneumophila. Journal of Bacteriology 161, 80-84. FIELDS,B. S., BARBAREE, J. M., SHOTTS,E. B., FEELEY, J. C., MORRILL, W., SANDEN, G. S. & DYKSTRA, M.J. (1986). Comparison of the guinea pig and protozoan models for determining virulence of Legionella species. Infection and Immunity 53, 553-559. GULIG,P. A. (1990). Virulence plasmids of Salmonella typhimuriumand other salmonellae. Microbial Pathogenesis 8, 3-1 1. HOLDEN,E. P., WINKLER,H. H., WOOD,D. 0. & LEINBACH, E. D. (1984). Intracellular growth of Legionella pneumophila within Acanthamoeba castellanii Neff. Infection and Immunity 45, 18-24. HORWITZ,M. A. & SILVERSTEIN, S. C. (1980). Legionnaires disease bacterium (Legwnella pneumophila) multiplies intracellularly in human monocytes. Journal of Clinical investigation 66, 441-450. ICHIGE,A., MATSUTANI, S., OISHI, K. & MIZUSHIMA,S. (1989). Establishment of gene transfer systems for and construction of the genetic map of a marine Vibrio strain. Journal of Bacteriology 171, 1825-1 834. JOHNSON, S. R. & SCHALLA, W. 0. (1982). Plasmids of serogroup 1 strains of Legionella pneumophila. Current Microbiology 7 , 143146. KADO,C. I. & LIU, S. T. (1981). Rapid procedure for detection and isolation of large and small plasmids. Journal of Bacteriology 145, 1365-1 373. KING,C. H., FIELDS,B. S., SHOTTS,E. B. & WHITE, E. H. (1991). Effects of cytochalasin D and methylamine on intracellular growth of Legionella pneumophila in amoebae and human monocyte-like cells. Infection and Immunity 59, 758-763. KNUDSON,G. B. & MIKESELL,P. (1980). A plasmid in Legionella pneumophila. Infection and Immunity 29, 1092-1095. J. F. & PARA,M. F. (1983). Plasmid profiles MAHER,W. E., PLOUFFE, of clinical and environmental isolates of Legionella pnewnophila serogroup 1. Journal of Clinical Microbiology 18, 1422-1423.
MANIATIS,T., FRITSCH,E. F. & SAMBROOK, J. (1982). Molecular Cloning: a Laboratory Handbook. Cold Spring Harbor, N Y : Cold Spring Harbor Laboratory. MARRA, A. & SHUMAN, H. A. (1989). Isolation of a Legionella pneumophila restriction mutant with increased ability to act as a recipient in heterospecific matings. Journal of Bacteriology 171, 2238-2240. MAURELLI, A. T. & SANSONETTI, P. J. (1988). Genetic determinants of Shigella pathogenicity. Annual Review of Microbiology 42, 127-1 50. MIKESELL, P., EZZELL,J. W. & KNUDSON, G. B. (1981). Isolation of plasmids in Legionella pneumophila and Legionella-like organisms. Infection and Immunity 31, 1270-1272. MINTZ, C. S. & SCHUMAN, H. A. (1988). Genetics of Legionella pneumophila. Microbiological Sciences 5, 292-295. NEWSOME, A. L., BAKER,R. L., MILLER,R. D. & ARNOLD,R. R. (1985). Interactions between Naegleria fowleri and Legionella pneumophila. Infection and Immunity 50, 449-452. PEARLMAN, D., JIWA,A. H., ENGELBERG, N. C. & EISENSTEIN, B. I. (1988). Growth of Legionella pneumophila in a human macrophagelike (U937) line. Microbial Pathogenesis 5, 87-95. PISCHL, D. L. & FARRAND,S. K. (1983). Transposon-facilitated chromosome mobilization in Agrobacterium tumefaciens. Journal of Bacteriology 153, 1451-1460. S. L. & FALKOW, S. (1981). Characterization PORTNOY, D. L., MOSELY, of plasmids and plasmid associated determinants of Yersinia enterocolitica pathogenesis. infection and immunity 21, 75-78. PORTNOY,D. A., WOLF-WATZ,H., BOLIN,I., BREEDER,A. B. & FALKOW, S. (1984). Characterization of common virulence plasmids in Yersiniaspecies and their role in the expression of outer membrane proteins. Infection and Immunity 43, 108-1 14. G. & ROMERO, E. (1973). RIVA,S., FIETTA,A., BERTI,M., SILVESTRI, Relationships between curing of the F episome by rifampin and by acridine orange in Escherichia coli. Antimicrobial Agents and Chemotherapy 3, 456-462. ROWBOTHAM, T. J. (1986). Current views on the relationships between amoebae, legionellae and man. Israel Journal of Medical Sciences 22, 678-689. SILHAVY, T. J., BERMAN, M. L. & ENQUIST, L. W. (1984). Experiments with Gene Fusions. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory. TREVORS,J. T., ODDIE,K. M. & BELLIVEAU, B. H. (1985). Metal resistance in bacteria. FEMS Microbiology Reviews 32, 39-54. TULLY,M. (1991). A plasmid from a virulent strain of Legionella pneumophila is conjugative and confers resistance to ultraviolet light. FEMS Microbiology Letters 90, 43-48.
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Isolation and characterization of a conjugative plasmid from Legionella pneumophila CLIFFORD S. MINTZ,~* BARRYS. FIELDS^ and CHANG-HUA Zou' Department of Microbiology and Immunology, University of Miami School of Medicine, Miami, Florida 33I0 I , USA Respiratory Diseases Branch, Division of Bacterial and Mycotic Diseases, National Center for Infectious Diseases, Centers for Disease Control, Atlanta, Georgia 30333, USA (Received 2 January 1992; revised 2 March 1992; accepted I1 March 1992)
The conjugative properties of an indigenous 85 MDa plasmid (designated pCH1) from Legioneffapneumophifa were studied. To determine if pCHl was transmissible by conjugation, mating experiments were brformed between legionellaethat harboured pCHl and several plasmid-lessrecipients. Plasmid transfer was monitored by colony hybridization, using a cloned 21.0 kb SalI restriction fragment from pCHl as a probe. The results from these experiments showed that pCHl could be conjugatively transferred into several strains of L. pneumophifa serogroup 1 but not into strain Bloomington-2 (serogroup 3) or Escherichia cofi. Southern hybridization experiments in which pCHl DNA was used as a probe showed that pCHl does not share homology with other indigenousL.pneumophifaplasmids. There was no detectable DNA homology between pCHl and L.pneumophifa chromosomal DNA. Additional mating experiments revealed that pCHl was unable to mobilize the L. pneumophifachromosome. The conjugative transfer of pCHl into plasmid-less avirulent or virulent serogroup 1strains did not alter the intracellular growth characteristicsof these strains in U937 cells, a human-monocytelike cell line, or in the amoeba Hartmanneffavermiformis.These results suggest that pCHl does not contribute to the ability of L. pneumophifato enter or grow within eukaryotic cells.
Int d u c tion Legionella pneumophila, the causative agent of Legionnaires' disease, is a facultative intracellular pathogen capable of entering and multiplying in cultured animal cells (Dreyfus, 1987; Pearlman et al., 1988), free-living amoebae (Fields et al., 1986; Holden et al., 1984; Rowbotham, 1986; Newsome et al., 1985) and human mononuclear phagocytes (Horwitz & Silverstein, 1980). At the present time, little is known about the bacterial factors that promote the intracellular lifestyle of this organism. Plasmids have been reported to carry genes that contribute to the virulence of a variety of Gram-negative intracellular pathogens including Yersinia spp. (BenGurion & Shafferman, 1981; Portnoy et al., 1981, 1984), Shigella spp. (Maurelli & Sansonetti, 1988) and Salmonella spp. (Gulig, 1990). Several groups have identified plasmids in both clinical and environmental isolates of
* Author for correspondence.Tel. (305) 547 6310; fax (305) 548 4623. Abbreviation: mAb, monoclonal antibody. 0001-7315 0 1992 SGM
L. pneumophila (Johnson & Schalla, 1982; Knudson & Mikesell, 1980; Maher et al., 1983; Mikesell et al., 1981). Early work indicated that the plasmids ranged in size from 23-85 MDa and did not confer an identifiable phenotype on strains that harboured them (Johnson & Schalla, 1982; Maher et al., 1983). Despite the presence of these plasmids in L. pneumophila, their contribution to the ability of L. pneumophila to enter and grow within eukaroytic cells has never been evaluated. Previous work from this laboratory (Mintz & Shuman, 1988) using broad host range IncP and IncQ plasmids showed that conjugation is possible in L. pneumophila. This finding, along with the identification of an 85 MDa plasmid in numerous L. pneumophila serogroup 1 clinical and environmental isolates (Schalla & Johnson, 1982; Maher et al., 1983) suggested that certain indigenous L. pneumophila plasmids may be transmissible by conjugation. In the present study, we evaluated the conjugative and virulence properties of an 85 MDa plasmid (designated pCH1) from L. pneumophila. Our results demonstrate that pCH 1 is self-transmissible by conjugation among serogroup 1 isolates of L. pneumophila. Also, pCHl does
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Table 1. Bacterial strains Abbreviations: Kmr, kanamycin resistant; Nal', naladixic acid resistant; Rip, rifampicin resistant; Sm', streptomycin resistant ; Trp-, tryptophan auxotroph; r-m+, restriction minus modification plus; r-m-, restriction minus modification minus. Designation L. pneumophila Philadelphia-1 Pontiac-1 NY 26 Ver 5 Rockport CH- 1 RH-1 UH-2 UPH-1 OLDA LPP2 LPP3 SF3256 SF3257 Albuquerque-1 Atlanta-1 Atlanta-4 AM511 CS267 CZ3001 Bloomington-2
cs2
CS302 Knoxville-1 CS323 E. coli DH5a
Relevant characteristics
Comments
Wild-type, clinical serogroup 1 isolate Clinical serogroup 1 isolate Clinical serogroup 1 isolate Clinical serogroup 1 isolate Clinical serogroup 4 isolate Environmental serogroup 1 isolate Environmental serogroup 1 isolate Environmental serogroup 1 isolate Environmental serogroup 1 isolate Clinical serogroup 1 isolate Environmental serogroup 1 isolate Environmental serogroup 1 isolate Environmental serogroup 1 isolate Environmental serogroup 1 isolate Clinical serogroup 1 isolate Clinical serogroup 1 isolate Clinical serogroup 1 isolate Smrr- m+ SmrRiPr-m+ AM511 : :TnSmob KMrSmr Clinical serogroup 3 isolate Serogroup 3 Smrt-m+ Serogroup 3 Sm'RiP Trp r-m+ Avirulent serogroup 1 clinical isolate Serogroup 1, SmrRiP
Plasmid-less Plasmid-less Plasmid-less Plasmid-less Plasmid-less Contains an 85 MDa plasmid Contains 45 and 85 MDa plasmids Contains a 45 MDa plasmid Contains a 33 MDa plasmid Contains an 85 MDa plasmid Contains an 85 MDa plasmid Contains 45 and 85 MDa plasmids Contains an 85 MDa plasmid Contains an 85 MDa plasmid Contains an 85 MDa plasmid Contains a 62 MDa plasmid Contains a 24 MDa plasmid Derived from Philadelphia-1 Spontaneous RiP mutant of AMSII Plasmid-less Plasmid-less, spontaneous Smr mutant of Bloomington-2 Derived from CS2 Plasmid-less Derived from Knoxville-1
NaP r- m+
Source*
H. Horwitz R. Miller M. Horowitz
1
J. F. Plouffe M. Horwitz
R. Miller J
A. Marra This study This study M. Horowitz This study This study R. Miller This study
C. Collins
* Addresses: M. Horwitz, UCLA Medical School, Los Angeles,CA, USA ;R. Miller, Universityof LouisvilleSchool of Medicine, Louisville, KY, USA; J. F. Plouffe, Ohio State University, Columbus, OH, USA; A. Marra, Massachusetts Institute of Technology, Boston, MA, USA; C. Collins, University of Miami School of Medicine, Miami, FL, USA. not contribute to the ability of L. pneumophila to enter or grow within eukaryotic cells.
Methods Bacterial strains and growth media. The bacterial strains used in this study are listed in Table 1. L. pneumophila was routinely grown in Albumin Yeast Extract (AYE) broth and on ACES-buffered Charcoal Yeast Extract (ABCYE) agar plates at 37 "C as previously described (Mintz & Shuman, 1988). Strains of Escherichia coliwere grown at 37 "C on L-agar or in L-broth. When necessary, the following antibiotic concentrations were used : streptomycin (Sm), 50 pg m1-I ; rifampicin (Rif), 20 pg ml-l; kanamycin (Km), 25 pg ml-l; ampicillin (Ap), 50 pg ml-l and tetracycline (Tc), 10 pg ml-l. Isolation and analysis of plasmid DNA. Plasmid DNA was isolated from L. pneumophila using the rapid method of Kado & Liu (1981) or the alkaline lysis protocol of Birnboim & Doly (1979). In some experiments, chromosomal DNA was removed from plasmid-containing lysates by centrifugationthrough ethidium bromide/CsClgradients (Maniatis et al. 1982). Plasmid DNA prepared by the Kado & Liu method was not cut by restriction endonucleases so plasmid DNA isolated by the Birnboim & Doly method was used for restriction endonuclease analysis in this investigation. Plasmid DNA and restriction enzyme digestions of plasmid DNA were analysed by
horizontal agarose gel electrophoresis using 0.4% and 0.7% agarose (Bio-Rad) gels. CloningofpCHl DNA. CsC1-purified pCHl DNA was digested with the restriction enzyme SalI (Promega), ligated with SalI-cleaved, alkaline-phosphatase-treated pBR322 DNA and transformed into CaC1,-treated E. coli DH5a. Transformants were selected on L-agar supplemented with ampicillin and putative recombinants were identified by their AprTcs phenotypes. The presence of pCHl sequences in recombinant plasmids was confirmed by Southern hybridization using 32P-labelledpCHl DNA as a probe. Mating experiments. Donor strain CH-1 (which harbours pCH 1 and contains no antibiotic-resistance markers) and antibiotic-resistant plasmidless recipients were grown to early exponential phase in AYE broth at 37 "C. Equal numbers of donors and recipients were incubated together on non-selectiveABCYE plates at 37 "C for 18 h as described by Mintz & Schuman (1988). After incubation, cells were removed from the plates, serially diluted in M63 salts buffer (Silhavy et al., 1984) and spread onto ABCYE agar plates that contained appropriate antibiotics (Sm, Rif or Km) to counterselect the donor strain CH-1. In some matings, strains that contained two or more drug markers were used as recipients. In these experiments, ABCYE agar that contained two antibiotics was used to counterselect CH-1. The plates were incubated at 37 "C. Colonies were replica-plated onto nitrocellulose filters, lysed with NaOH, probed with 32P-labelledpBR322 DNA (containing a 21.0 kb SalI fragment from pCH1) and subjected to
Conjugativeplasmid from Legwnella pneumophila autoradiography. We routinely screened approximately 2000-3000 transconjugants for the presence of pCHl DNA. Preliminary experiments showed that 32P-labelled pBR322 DNA by itself did not hybridize with pCHl or L. pneumophila chromosomal DNA. Hybridizations were performed under conditions of high stringency (Silhavy et al., 1984). Colonies that hybridized with the probe were picked from master plates and purified twice by passage on selective media. Plasmid DNA was isolated from these colonies, digested with SalI and examined by agarose gel electrophoresis. All mating experiments were repeated at least two times. For mating experiments involving DNAase treatment, equal numbers of donors and recipients were incubated for 18 h at 37 "C on ABCYE agar that contained 1 mg DNAase ml-l. Cells were then removed from the plates and spread onto antibiotic-containing ABCYE agar as described above. Chromosome mobilization experiments using strain CH-1 as a donor and auxotrophic mutants of strains Bloomington-2 (Gua- and Trp-) and Philadelphia-1 (Thy- and Trp-) as recipients were done according to Mintz & Schuman (1988). Colony immunoblast assay. Single colonies of donors, recipients and transconjugants were inoculated onto 0.45 pm nitrocellulose filters (Fisher) placed on ABCYE agar plates and incubated for 24-48 h at 37 "C. Filters were removed from the plates and incubated in Trisbuffered saline (TBS; 50 mM-Tris/HCl, 150 mM-NaCl, pH 7.5) containing 5 % (w/v) nonfat dried milk (Carnation) for 2 h at room temperature. The filters were washed several times with TBS and incubated with monoclonal antibody (mAb) 1E6 (1 : 1000dilution) on a rotary shaker (New Brunswick) for 1 h at room temperature. mAb 1E6 (kindly provided by W. Johnson, University of Iowa, USA) is specific for serogroup 1 lipopolysaccharide. After incubation, the filters were washed several times with TBS and probed with horseradishperoxidase-conjugated goat-antimouse IgG antibodies (1 : 1000 dilution, Cappel) for 1 h at room temperature. After a series of four washes with TBS, filters were immersed in a solution of 0.05% 4-chloro-lnaphthol (Sigma) and 0.015 % hydrogen peroxide. Antibiotic and heavy metal resistance. The ability of pCH 1 to confer resistance to antibiotics or heavy metals was tested in the following manner. Single colonies of isogenic plasmidless and pCH 1-containing strains of L. pneumophila were inoculated onto ABCYE agar and ABCYE agar containing a specific antibiotic or heavy metal. The plates were incubated at 37 "C for 5 d. Resistance to the following heavy metals was tested using the concentrations suggested by Trevors et al. (1985): cadmium chloride (1 m~), sodium arsenate (38 mM), cobalt chloride (3 mM), copper sulphate (20 mM), lead nitrate (0.3 mM), nickel chloride (3 mM), zinc chloride (1 mM) and silver nitrate (5 mM). Antibiotics were tested at the concentrations mentioned above. Infection of U937 cells and Hartmannella vermiformis with L. pneumophila. Human-monocyte-like cells (U937) were infected with legionellae as previously described (King et al., 1991). U937 cell monolayers were grown in RPMI 1640 (Cellgro) plus 20% (v/v) normal human serum for up to 3 d at 37 "C in 5 % (v/v) C02.At daily intervals, samples were removed from the infected monolayers, diluted in M63 salts buffer and plated on ABCYE agar to determine numbers of L. pneumophila. Plate-grown legionellae were co-cultured with the amoeba H . vermiformis at 35 "C for 7 d according to King et al. (1991). At various times, samples were removed from the co-cultures, serially diluted in buffer, and plated on ABCYE agar to determine the numbers of L. pneumophila. Previous studies using U937 cells (King et al., 1991; Pearlman et al., 1988) and H. vermiformis (King et al., 1991) demonstrated that any increase in numbers of L. pneumophila as measured above represent intracellular growth of the bacterium.
138 1
Isolation and characterization of pCHl The plasmid content of five serogroup 1 strains (CH-1, OLDA, Albuquerque 1, LpP, and SF3256) reported to carry 85 MDa plasmids was analysed by agarose gel electrophoresis. Each strain contained a single plasmid and the electrophoretic mobility of the plasmids was identical (data not shown). Digestion of each plasmid with SalI or BamHI yielded identical fragment patterns (data not shown). These results suggested that strains CH-1, OLDA, Albuquerque-1, LpP, and SF3256 harboured the same plasmid, subsequently designated pCH1. Restriction endonuclease analysis of pCH 1 revealed that EcoRI, Hind111or HaeIII generated 2 13 restriction fragments. Digestion with BglI, PvuII, SalI, BglII or NheI yielded 5-7 restriction fragments (data not shown). pCH1 was not cleaved by NotI, SI', ApaI or RsrII. We also determined that pCHl has a single BarnHI site contained within the 11 kb SalI restriction fragment (Fig. 1). Attempts to construct a detailed restriction map of pCHl were hampered by the large number of restriction fragments generated during single and double digests and our inability to clone the majority of the plasmid genome in E. coli (see below). Pulse-field gel electrophoresis of BamHI-digested plasmid DNA showed that the apparent molecular size of pCH 1 is 125130 kb (data not shown). This is in agreement with the predicted molecular size of pCH1, which was previously determined to be 85 MDa (128 kb). The results from antibiotic sensitivity tests indicated that pCH1 did not encode resistance to Rif, Sm, Ap, Km or Tc. Neither did the plasmid confer resistance to any of the heavy metals tested. Attempts to cure pCH1 from plasmid-bearing strains by treatment with acridine orange (Riva et al., 1973), ethidium bromide (Bouanchaud et al., 1969), growth at 41 "C or by repeated subculture on ABCYE agar at 37 "C were unsuccessful. This suggested that pCH1 was stably maintained in L. pneumophila in the absence of obvious selective pressure. Cloning of pCHl DNA As shown in Fig. 1, the restriction enzyme SalI cleaves pCH1 DNA into six distinct restriction fragments. We attempted to isolate and clone each of the six SalI fragments from pCH1. Despite repeated attempts using either pBR322 or pACYC184 as cloning vectors, we were only able to isolate the 21.0 kb SalI fragment from pCH1. This fragment was cloned into the unique SalI site of pBR322 (Fig. 1) and subsequently used as a probe in plasmid transfer experiments.
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Fig. 1 . Ethidium-bromide-stained 0.4% agarose gel that contains the cloned 21.0 kb SalI fragment of pCH1. Lanes : A, 1 Hind111 molecular size standards; B, undigested pCHl DNA; C, pCHl DNA digested with SalI; D, pBR322 DNA that contains the cloned fragment digested with SalI; E, pBR322 DNA digested with SalI. Arrow indicates the position of the 21.0 kb fragment of pCH1.
Conjugative transfer of pCHl
To determine whether pCHl is conjugative, mating experiments between strain CH-1 (which harboured pCH 1) and several antibiotic-resistant plasmid-less L. pneumophila recipients were done. Since pCHl does not encode any selectable markers, we assessed the ability of strain CH-1 to conjugally transfer pCH1 by colony hybridization using the cloned 21 kb SalI fragment from pCHl as a probe. Initial mating experiments, in which strain AM51 1 was used as the recipient, revealed that several colonies on the selection plates hybridized with the pCHl probe. The electrophoretic profile of SalI-digested plasmid DNA from each of these colonies was identical to that of SalI-digested pCH1 DNA (data not shown). This suggested that pCH1 was transferred by a mating
process. However, the possibility existed that colonies that contained pCH 1 were spontaneous antibioticresistant mutants of strain CH-1 rather than actual transconjugants. To eliminate this possibility, we did additional mating experiments using multiple-antibioticresistant recipients in which two antibiotics were used to counterselect donor strain CH- 1. The frequency of spontaneous mutation to resistance for two antibiotics for strain CH-1 would be approximately lo-' *. However, pCH 1 was detected in exconjugants at frequencies ranging from 10-3-10-4 per recipient. These results supported the idea that colonies that contained pCHl were transconjugants rat her than anti biotic-resistant mutants of strain CH-1. This was confirmed by colony immunoblot experiments with putative transconjugants using mAb 1E6, which does not bind to strain CH-1 but is reactive with each of the other serogroup 1 strains used as recipients in this study. In all cases, colonies that hybridized with the pCHl probe also bound mAb lE6 (data not shown). This demonstrated that pCHl was transferred from strain CH-1 to L. pneumophila recipients during the mating process. The detection of pCH 1 in transconjugants obtained from matings in the presence of DNAase (1 mg ml-I) provided conclusive evidence that pCH l was selftransmissible by conjugation (data not shown). A summary of the mating experiments done in this study is presented in Table 2. By determining the number of pCH1-containing colonies among the total number of transconjugants screened, we estimated that pCH 1 was transferred at frequencies ranging from l 0-3- 10-4 per recipient. Of interest, pCHl could only be conjugally transferred into serogroup 1 recipients. We were unable to detect transfer of pCHl into a serogroup 3 recipient (strain Bloomington-2) or E. coli DHSa. Additional mating experiments between strain CH- 1 and several serogroup 1 or serogroup 3 auxotrophic recipients showed that pCH1 could not promote the chromosomal transfer of the gua, thy or trp loci in L. pneumophila (data not shown). pCHI does not share homology with L. pneumophila chromosomal DNA or other indigenous plasmids
To determine if pCH1 shared DNA homology with other L. pneumophila plasmids or with L. pneumophila chromosomal DNA, we performed colony hybridization experiments with plasmid-less and plasmid-containing strains of L. pneumophila. In these experiments, CsC1-purified pCH1 DNA labelled with 32Pwas used as a probe. pCH1 DNA did not hybridize with strains of L. pneumophila that contained indigenous plasmids other than pCHl (strains UH-2, UPH-1, Atlanta4 and Atlanta-4; Fig. 2). This suggested that pCH1 does not share detectable
Conjugative plasmid from Legionella pneumophila
I 38 3
Fig. 2. Colony hybridization experiments with plasmid-less and plasmid-bearing strains of L. pneumophila. Individual colonies from plasmid-lessand plasmid-bearing strains of L.pneumophila were patched onto nitrocellulosefilters,treated with NaOH and probed with 32P-labeHedCsC1-purified pCHl DNA. Colonies that hybridized with the probe were visualized by autoradiography. Only those strains that harboured an 85 MDa plasmid hybridized with the pCHl probe.
Table 2. Summar? of mating experiments Abbreviations, see Table 1. Recipient Donor
Strain
CH-1 CH-1 CH-1 CH-I CH-1 CH-1 CH-1
AM51 1 CS267 CZ3001 CS320 CS2 CS302 DH5a
Phenotype
Selection
Plasmid transfer detected
Philadelphia-1 serogroup 1 Smrr-m+ Philadelphia-1 serogroup 1 SmrRiPr-m+ Philadelphia-1 serogroup 1 : :Tn5mb KmrSmrr-m+ Knoxville-I serogroup 1 Srn'RiP Bloomington-2 serogroup 3 Sm'r-mBloomington-2 serogroup 3 RiP Trp-r-mEscherichia coli Nalrr-m+
Sm Sm Rif Km Sm Sm Rif Sm Rif Nal
Yes Yes Yes Yes No No No
DNA homology with other Legionella plasmids. In contrast, all of the L. pneumphila strains that contained an 85 MDa plasmid hybridized with the probe (LpP2, LpP,, OLDA, RH-1, SF3256 and SF3257; Fig. 2).
The plasmid-less serogroup 1 strains Philadelphia-1, Knoxville-1 and UH-1 did not hybridize with pCH1 DNA (Fig. 2). Also, the plasmid-less serogroup 3 strain, Bloomington-2, did not hybridize with pCH 1.
1384
1
C. S . Mintz, B. S . Fields & C.-H. Zou
.,
0
1
2
1 3 0 Time post-infection (d)
1
2L
Fig. 3. Infection of U937 cell monolayers with isogenic plasmid-less and plasmid-bearing strains of L. pneumophila. U937 cell monolayers were infected with L. pneumophila according to the methods of King etul. (1991). (a) AM51 1 ; 0 ,AM51 l(pCH1). @)A,CS323; A, CS323(pCHl). Each point represents the mean f SE for three separate U937 cell cultures.
Additional experiments demonstrated that pCH 1 DNA also did not hybridize with the plasmid-less serogroup 1 strains Pontiac-1, NY 26 and Ver 5, or the plasmid-less serogroup 4 strain Rockport (data now shown). This suggests that these strains do not contain chromosomal sequences homologous with pCHl DNA. pCHl does not aflect the intracellular growth of L . pneumophila
Using the mating procedure outlined above, we introduced pCHl into strains AM51 1 and CS323 to construct isogenic pairs of plasmid-less and plasmidcontaining strains. Strain AM5 11 is virulent and capable of intracellular growth (Marra & Shuman, 1989). In contrast, strain CS323 is avirulent and incapable of intracellular multiplication (C. S. Mintz, unpublished). Both isogenic sets were used to infect monocyte-like U937 cells or the amoeba Hartmannella vermiformis. pCHl did not affect the ability of strain AM511 to multiply in U937 cells (Fig. 3a) or in H. vermiformis(data not shown). Moreover, the plasmid did not restore the ability of the avirulent strain CS323 to replicate in either U937 cells (Fig. 3b) or in H. vermiformis (data not shown).
Discussion In this study, we have shown that pCH1, an indigenous L. pneumophila plasmid, is self-transmissibleby conjugation among serogroup l strains of L . pneumophila. Prior
to the present study, we (Mintz & Shuman, 1988) as well as others (Dreyfus & Iglewski, 1985; Chen et al., 1986) demonstrated that broad host range plasmids of the IncP and IncQ incompatibility groups could easily be transferred via conjugation between different L. pneumophila isolates. Recently, Tully (1991) demonstrated that a 36MDa plasmid from L . pneumophila strain Dodge (serogroup 1) could be transferred by conjugation to other serogroup 1 strains. Our results, along with those of Tully (199I), suggest that conjugation mediated by indigenous plasmids may be a mechanism of genetic exchange for L. pneumophila. To date, neither transformation nor transduction has been reported for L. pneumophila. Despite repeated attempts, we were unable to detect conjugative transfer of pCHl into a serogroup 3 strain (Bloomington-2) of L. pneumophila. This was somewhat surprising since strain Bloomington-2 lacks the LpnII restriction-modification system of L . pneumophila (Marra & Shuman, 1989), and is an excellent recipient in homologous and heterospecific matings involving IncP and IncQ plasmids (Chen et al., 1984; Mintz & Shuman, 1988). It is not clear why we were unable to detect transfer of pCHl in strain Bloomington-2. It is possible that pCH1 cannot be stably maintained in this strain. Experiments are underway to determine if pCH1 can be transferred by conjugation into strains from other serogroups of L . pneumophila. pCHl could not be transferred from strain CH-1 into E. coli strain DH5a. Since the 36 MDa plasmid described by Tully (1991) could also not be transferred from serogroup 1 strains of L. pneumophila into E. coli or Pseudomonas aeruginosa, it would seem that conjugative L . pneumophila serogroup 1 plasmids have a relatively narrow host range. Colony hybridization experiments revealed that pCH 1 did not share detectable DNA homology with the 24,33, 45 or 62 MDa plasmids harboured by certain serogroup 1 strains of L. pneumophila, and that pCHl was not related to these other L. pneumophila plasmids (Fig. 2). Therefore, it is likely that pCHl belongs to a different incompatibility group than these other L . pneumophila plasmids. In support of this idea, the 45 MDa plasmid has been found in two serogroup 1 strains that also harbour pCH1, viz. RH-1 (Maher et al. 1983) and LpP3 (C. S. Mintz, unpublished). Attempts to assign pCHl to a known incompatibility group have been hampered by the lack of a selective marker on pCH 1 and the difficulty associated with introducing plasmid DNA into L. pneumophila. Recently, we determined that electroporation is an efficent way of delivering plasmid DNA into L. pneumophila (C. S. Mintz & C.-H. Zou, unpublished). This should permit future experiments designed to identify the incompatibility group of pCHl. It is well-establishedthat the transfer of chromosomal
Conjugative plasmid from Legionella pneumophila
genes during conjugation results from integration of a conjugative plasmid, such as F, into the donor chromosome (Deich & Green, 1987; Silhavy et al., 1984). Integration of plasmid DNA into the host chromosome usually occurs by recombination between homologous DNA sequences contained on the plasmid and the chromosome (Bartowsky et al., 1987; Brenton et al., 1985). As previously noted, several plasmid-less L. pneumophila strains failed to hybridize with the pCH1 probe in colony hybridization experiments. These results indicate that there is no detectable DNA homology between the L. pneumophila chromosome and pCH1. This finding could explain the inability of pCH1 to mobilize the L. pneumophila chromosome. In the absence of DNA sequence homology, pCH1 would be unable to integrate into the L. pneumophila chromosome. Consequently, the unintegrated plasmid would be incapable of promoting the transfer of chromosomal markers. Of interest, Bartowsky et al. (1987) suggested that the lack of chromosome mobilizing activity exhibited by a conjugative plasmid from Vibrio cholerae was due to the lack of significant homology between the V . cholerae chromosome and the plasmid. Thus, although pCHl is selftransmissible by conjugation, it is not capable of promoting detectable chromosomal gene transfer in L. pneumophila. Virulence plasmids are essential for the pathogenicity of a variety of intracellular pathogens including Salmonella spp. (Gulig, 1990), Shigella spp. (Maurelli & Sansonetti, 1988) and Yersinia spp. (Portnoy et al., 1981, 1984). In the light of these observations, we evaluated the contribution of pCHl to the ability of L. pneumophila to multiply intracellularly in eukaryotic cells. The introduction of pCH1 into the plasmid-less, virulent strain AM51 1 did not augment or alter the ability of this strain to grow within U937 cells or H. vermiformis. Moreover, the presence of pCHl in the avirulent strain CS323 did not restore its ability to multiply in U937 cells or H. vermiformis. These results indicate that pCHl does not encode factors thzt contribute to the intracellular growth of L. pneumophila within eukaryotic host cells. Our results are consistent with the findings of Bollin et al. (1985) who compared the ability of a plasmid-less and plasmid-containing serogroup 1 strain to infect guinea pigs following intreperitoneal injection. The plasmidcontaining strain harboured pCH1 and a 45 MDa plasmid. Their results showed that there was no statistically significant difference in the IDs0 of the plasmid-less or plasmid-bearing strain. Interestingly, the LDS0of the plasmid-less strain was significantly lower (approximately 10-fold) than that of the plasmidcontaining strain. However, it is important to note that the plasmid-less and plasmid-containing strains used in this study were not isogenic. Therefore, it is difficult to
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attribute the reduced virulence exhibited by the plasmidcontaining strain to the presence of pCHl or the 45 MDa plasmid. Nonetheless, our results suggest that pCH1 is not required for expression of virulence by L. pneumophila. Although pCHl cannot mobilize the L. pneumophila chromosome,the self-transmissiblenature of the plasmid suggests that it may contribute to the exchange of genetic information among serogroup 1 isolates. For example, the acquisition by pCHl of transposons which code for antibiotic resistance could pose serious problems in the treatment of Legionnaires’ disease. Moreover, the presence of transposons on pCH 1 could facilitate integration of the plasmid into the L. pneurnophila chromosome by transposon-mediated recombination (Ichige et al., 1989, Pischl & Farrand, 1983). This, in turn, could promote Hfr-like chromosomal gene transfer in L. pneumophila. In support of this notion, we have previously demonstrated that conjugative plasmids that contain transposons can be used to mobilize the L. pneumophila chromosome (Mintz & Shuman, 1988). At the present time, it is not clear what functions are encoded by pCH 1. It does not encode resistance to any of the antibiotics or heavy metals tested nor does it contribute to the intracellular growth of L. pneumophila. The absence of pCHl in many environmental and clinical serogroup 1 isolates suggests that any function(s) encoded by the plasmid are not essential for the survival of L. pneumophila in aquatic environments or in the infected host. We thank M. Horwitz, R. Miller, J. Plouffe and C. Collins for supplying us with strains used in this study. This work was supported by a research grant awarded to C. Mintz from the American Lung Association of Florida.
References BARTOWSKY, E. J., MORELLI, G., KAMKE, M. & MANNING, P. (1987). Characterization and restriction analysis of the P sex factor and the cryptic plasmid of Vibrio cholerae strain V58. Plasmid 18, 1-7. BEN-GURION,R. & SHAFFERMAN, A. (1981). Essential virulence determinants of different Yersinia species are carried on a common plasmid. Plasmid 5, 183-187. BIRNBOIM, H. C. & DOLY,J. (1979). A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Research 7 , 1513-1523. BOLLIN,C. E., PLOUFFE, J. F., PARA,M. F. & PRIOR,R. B. (1985). Difference in virulence of environmental isolates of Legionella pneumophila. Journal of Clinical Microbiology 21, 674-677. BOUANCHAUD, D. H., SCAVIZZI, M. R. & CHABBERT, Y. A. (1969). Elimination by ethidium bromide of antibiotic resistance in enterobacteria and staphylococci.Journal of General Microbiology 54, 417-425. BRENTON, A. M., JAOUA, S. & GUESPIN-MICHEL, J. (1985). Transfer of plasmid RP4 to Myxococnts xanthus and evidence for its integration into the chromosome. Journal of Bacteriology 161, 523-528.
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CHEN,G. C. C., LEMA,M. & BROWN,A. (1984). Plasmid transfer into members of the Legionellaceae. Journal of Infectious Diseases 150, 5 13-5 16. CHEN, G. C. C., BROWN,A. & LEU, M. W. (1986). Restriction endonuclease activities in the legionellae. Canadian Journal of Microbiology 32, 591-593. DEICH,R. A. & GREEN,B. A. (1987). Mobilization of Haemophilus infuenzae chromosomal markers by an Escherichia coli F factor. Journal of Bacteriology 169, 1905-1910. L. A. (1987). Virulence associated ingestion of Legionella DREYFUS, pneumophila by HeLa cells. Microbial Pathogenesis 3, 45-52. DREYFUS,L. A. & IGLEWSKI,B. H. (1985). Conjugation-mediated genetic exchange in Legwnella pneumophila. Journal of Bacteriology 161, 80-84. FIELDS,B. S., BARBAREE, J. M., SHOTTS,E. B., FEELEY, J. C., MORRILL, W., SANDEN, G. S. & DYKSTRA, M.J. (1986). Comparison of the guinea pig and protozoan models for determining virulence of Legionella species. Infection and Immunity 53, 553-559. GULIG,P. A. (1990). Virulence plasmids of Salmonella typhimuriumand other salmonellae. Microbial Pathogenesis 8, 3-1 1. HOLDEN,E. P., WINKLER,H. H., WOOD,D. 0. & LEINBACH, E. D. (1984). Intracellular growth of Legionella pneumophila within Acanthamoeba castellanii Neff. Infection and Immunity 45, 18-24. HORWITZ,M. A. & SILVERSTEIN, S. C. (1980). Legionnaires disease bacterium (Legwnella pneumophila) multiplies intracellularly in human monocytes. Journal of Clinical investigation 66, 441-450. ICHIGE,A., MATSUTANI, S., OISHI, K. & MIZUSHIMA,S. (1989). Establishment of gene transfer systems for and construction of the genetic map of a marine Vibrio strain. Journal of Bacteriology 171, 1825-1 834. JOHNSON, S. R. & SCHALLA, W. 0. (1982). Plasmids of serogroup 1 strains of Legionella pneumophila. Current Microbiology 7 , 143146. KADO,C. I. & LIU, S. T. (1981). Rapid procedure for detection and isolation of large and small plasmids. Journal of Bacteriology 145, 1365-1 373. KING,C. H., FIELDS,B. S., SHOTTS,E. B. & WHITE, E. H. (1991). Effects of cytochalasin D and methylamine on intracellular growth of Legionella pneumophila in amoebae and human monocyte-like cells. Infection and Immunity 59, 758-763. KNUDSON,G. B. & MIKESELL,P. (1980). A plasmid in Legionella pneumophila. Infection and Immunity 29, 1092-1095. J. F. & PARA,M. F. (1983). Plasmid profiles MAHER,W. E., PLOUFFE, of clinical and environmental isolates of Legionella pnewnophila serogroup 1. Journal of Clinical Microbiology 18, 1422-1423.
MANIATIS,T., FRITSCH,E. F. & SAMBROOK, J. (1982). Molecular Cloning: a Laboratory Handbook. Cold Spring Harbor, N Y : Cold Spring Harbor Laboratory. MARRA, A. & SHUMAN, H. A. (1989). Isolation of a Legionella pneumophila restriction mutant with increased ability to act as a recipient in heterospecific matings. Journal of Bacteriology 171, 2238-2240. MAURELLI, A. T. & SANSONETTI, P. J. (1988). Genetic determinants of Shigella pathogenicity. Annual Review of Microbiology 42, 127-1 50. MIKESELL, P., EZZELL,J. W. & KNUDSON, G. B. (1981). Isolation of plasmids in Legionella pneumophila and Legionella-like organisms. Infection and Immunity 31, 1270-1272. MINTZ, C. S. & SCHUMAN, H. A. (1988). Genetics of Legionella pneumophila. Microbiological Sciences 5, 292-295. NEWSOME, A. L., BAKER,R. L., MILLER,R. D. & ARNOLD,R. R. (1985). Interactions between Naegleria fowleri and Legionella pneumophila. Infection and Immunity 50, 449-452. PEARLMAN, D., JIWA,A. H., ENGELBERG, N. C. & EISENSTEIN, B. I. (1988). Growth of Legionella pneumophila in a human macrophagelike (U937) line. Microbial Pathogenesis 5, 87-95. PISCHL, D. L. & FARRAND,S. K. (1983). Transposon-facilitated chromosome mobilization in Agrobacterium tumefaciens. Journal of Bacteriology 153, 1451-1460. S. L. & FALKOW, S. (1981). Characterization PORTNOY, D. L., MOSELY, of plasmids and plasmid associated determinants of Yersinia enterocolitica pathogenesis. infection and immunity 21, 75-78. PORTNOY,D. A., WOLF-WATZ,H., BOLIN,I., BREEDER,A. B. & FALKOW, S. (1984). Characterization of common virulence plasmids in Yersiniaspecies and their role in the expression of outer membrane proteins. Infection and Immunity 43, 108-1 14. G. & ROMERO, E. (1973). RIVA,S., FIETTA,A., BERTI,M., SILVESTRI, Relationships between curing of the F episome by rifampin and by acridine orange in Escherichia coli. Antimicrobial Agents and Chemotherapy 3, 456-462. ROWBOTHAM, T. J. (1986). Current views on the relationships between amoebae, legionellae and man. Israel Journal of Medical Sciences 22, 678-689. SILHAVY, T. J., BERMAN, M. L. & ENQUIST, L. W. (1984). Experiments with Gene Fusions. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory. TREVORS,J. T., ODDIE,K. M. & BELLIVEAU, B. H. (1985). Metal resistance in bacteria. FEMS Microbiology Reviews 32, 39-54. TULLY,M. (1991). A plasmid from a virulent strain of Legionella pneumophila is conjugative and confers resistance to ultraviolet light. FEMS Microbiology Letters 90, 43-48.
